The general objective of this research project is to understand the mechanism of DNA replication. The main experimental approach being used is to examine discontinuous DNA synthesis in vitro, using lysates of E. coli coated as films on cellophane discs (the "Bonhoeffer system"). We have two specific goals: 1) To define the origin, structure and function of discontinuously synthesized DNA intermediates that are detected during replication; 2) to obtain a more fundamental understanding of the specificity of the enzymes involved in DNA replication, with a particular reference to their processivity. We have recently been able to estimate the frequency of true initiation events (i.e., the fraction of the truly discontinuously synthesized DNA intermediates) and the frequency of uracil excision events at the replication fork. Both appear to lead to the formation of "Okazaki pieces." If the in vitro system is a reasonable monitor of the events that are going on in vivo, these results suggest that 70-80% of 5' termini of Okazaki pieces arise by uracil excision, and only 20-30% arise by true initiation mechanisms. Furthermore, all of the initiation events take place on only one strand of the replication fork. We will continue these studies in the coming year.